Our studies during the past year have centered on the definition of spontaneous immunosuppression which occurs in patients with advanced cancer. Of the population studied, response of 6/14 melanoma patients was significantly depressed. Those patients displaying the presence of immunosuppressive serum components responded less favorably to treatment than those patients with normal serum values. We have developed a reliable assay of immunosuppression of phytohemagglutinin-P (PHA) induced blastogenesis by serum from selected melanoma patients, using normal human lymphocytes isolated by sedimentation and cotton column adherence. These lymphocyte cultures were found to contain both responder cells (subpopulation x) and regulator cells (subpopulation y). Lymphocytes isolated by gradient centrifugation on sodium metrizoate-Ficol contained responder cells (x) but were considerably depleted in regulator cells (y). Cultures isolated by this method were stimulated by PHA but were not suppressed by the addition of melanoma patient serum. When lymphocytes were isolated by a cotton column adherence/ Lymphoprep centrifugation-double separation, subpopulations (x) and (y) were isolated. We have established that both subpopulations are necessary for suppression to occur, and that (y) operates as the regulator of (x). Finally, by manipulating B cell and T cell populations isolated by nylon column adherence or 2-aminoethylisothiouronium bromide hydrobromide (AET) rosette separation, it appears that the regulator ability of subpopulation (y) is the result of B cell activation of suppressor T cells.